In recent years, presence or absence of mutation in a particular gene has been emphasized in various fields. For example, in DNA identification in forensics, determination of presence or absence of one nucleotide substitution by mutation or polymorphism in DNA is already well known as an individual identification technique. In addition, also in the medical field, correlation between specific genetic polymorphisms and drug susceptibility has become known, and trials to reduce the risk of drug-induced health disaster by investigating specific genetic polymorphisms have been done. Moreover, the detection of mutation in the gene is also employed for detection etc. of a mutant-type DNA holder of a hereditary disorder and its importance is increasing rather than before.
As for the conventional simple method for detecting single nucleotide substitution in DNA, SSCP method has been known. The SSCP method is a method through the use of a fact that DNA fragment is amplified by polymerase chain reaction (PCR) method, and the amplified product is made to the single-stranded DNA by thermal melting, and in the cooling process after thermal melting, said single-stranded DNA forms base-pairs partially within a molecule; and in this method, the molecule having single nucleotide mutation is changed to the shape of the single-stranded DNA, and based on the difference of mobility of electrophoresis from a wild-type DNA, separation and discrimination of the mutant-type DNA is performed. However, to maintain the molecular shape of single-strand DNA, this SSCP method needed to keep the temperature constant during electrophoresis. Therefore, the electrophoresis equipment is required to be provided with a circulation type constant temperature system, and remained a problem that a big unit would be needed.
And, as a method for solving the above-described problem on the SSCP method, a loop hybrid (LH) method in which single-stranded oligo DNA is added to the reaction solution after the PCR reaction of DNA fragment to hybridize with DNA fragment, and the mutant-type DNA is discriminated by electrophoresis on the basis of the structural difference of the obtained hybrid, has been proposed (JP-A-2007-61080).    Non-patent Literature 1: Orita, M. et al., Genomics 5, 874-879 (1989);    Non-patent Literature 2: Matsukuma, S. et al., J. Mol. Diag. 8, 504-512 (2006);    Patent Literature 1: JP-A-2007-61080.